Abstract The endoplasmic reticulum (ER) is a major site for protein folding and maturation within the cell, and a wide array of human diseases, including neurodegenerative diseases, familial protein folding disorders, and diabetes, are associated with disruptions to ER protein folding homeostasis. Endoplasmic reticulum associated degradation (ERAD) is a highly conserved pathway that functions to promote protein homeostasis by preventing misfolded protein accumulation. It is an integral part of the ER unfolded protein stress response. In addition to degradation of misfolded proteins, ERAD regulates the protein levels of ER-resident enzymes, such as the HMG-CoA reductase, the rate-limiting enzyme in sterol synthesis. Interestingly, the ERAD machinery is hijacked by viral pathogens during infection, meaning it is a potential therapeutic target. The process of ERAD involves transferring target protein substrates from the ER lumen or membrane to the cytosol for degradation. It can be divided into five distinct steps: substrate recognition, retro-translocation across the ER membrane, polyubiquitination, extraction from the membrane, and proteasomal degradation. While the molecular details of the later steps have become increasingly clear, how ERAD machinery recognizes misfolded or other substrate targets remains ambiguous. Previous work identified substrate glycosylation state as an important influencer of interactions with ERAD machinery. Nevertheless, glycosylation is dispensable for degradation, while substrate misfolding is not. The aims of this proposal seek to identify principles of substrate recognition by ERAD and other protein quality control machinery in the secretory pathway. Combining DNA sequencing technology and cell sorting techniques, we will generate and screen libraries of mutated or degron-fused non-ERAD substrates in S. cerevisiae to identify features that are recognized by ERAD machinery. We will then validate features through cell biology and in vitro reconstitution assays. Understanding the principles of substrate recognition by ERAD will illuminate the physiological ERAD targets, should allow us to predict additional targets in higher organisms, and understand exploitation of the system by certain pathogens.